Figure 1: Ox-LDL induces VSMC-derived foam cell death, causes mtROS accumulation, reduces ΔΨm, and upregulates pro-atherosclerosis factor expression. VSMCs were treated for 72 h with 5 µg/mL cyclodextrin-cholesterol complex to establish VSMC-derived foam cells, which were subsequently treated with different concentrations of ox-LDL for 24 h. (a) Viability of VSMC-derived foam cells was measured using the CCK8 assay. (b) LDH release was evaluated using a cytotoxicity detection kit. (c) Mitochondrial ROS levels were detected using the MitoSOX™ Red mitochondrial superoxide indicator after treating VSMC-derived foam cells for 6 h with different concentrations of ox-LDL. (d) A JC-1 staining kit was used to measure mitochondrial membrane potential after treating VSMC-derived foam cells for 6 h with different concentrations ox-LDL. (e) Real-time PCR analysis was performed to measure the mRNA levels of atherosclerosis-related factors, namely, MYH11, CD36, CD47, ICAM-1, VCAM-1, IL-1β, IL-18, MCP-1, MMP9, and PCSK9. Data are presented as mean ± SD of three independent experiments. *p < 0.05 vs. the control group; **p < 0.01 vs. the control group.

3.2. Ox-LDL (10 μg/mL) Induces VSMC-Derived Macrophage-Like Foam Cell Necroptosis

Local inflammation in plaques plays an important role in atheroma progression. Because apoptotic and necroptotic cell death have distinct effects on inflammation, the type of cell death induced by ox-LDL needs to be determined. To illustrate the pattern of ox-LDL-induced cell death, cell morphology in each treatment group was examined using electron microscopy. The appearance of plasma membrane destruction and organelle fragmentation or swelling in 10 μg/mL ox-LDL-treated VSMC-derived macrophage-like foam cells revealed that the cells were necrotic (Fig. 2a). We used Annexin V and PI staining to determine the cell death rate (Fig. 2b). VSMC-derived macrophage-like foam cells without any treatment exhibited cell death, and PI-positive cells accounted for a major portion of these cells. As PI uptake is an indicator of the loss of cell membrane integrity, according to the feature of necroptosis cells, we calculated PI-positive cell rate and found that 10 μg/ mL ox-LDL treatment significantly promoted VSMC-derived macrophage-like foam cell necroptosis (Fig. 2c).